Kv1.2 Knock out mouse
Voltage gated potassium channel Kv1.2 a class of delayed rectifying potassium channels which allow nerve cells to efficiently repolarize by allowing intracellular potassium to outside the cell 1. Kv1.1 and Kv1.2 are the two most abundant Kv1 subunit types and are commonly in the same tetramers found in brain along with several different combinations of Kv1.1, Kv1.2, Kv1.3, Kv1.4, Kv1.6 channels making it hard to charecterize the roles played by these different channels. One strategy to study them involves gentic deletion (knock out) of these channels. Generation of Kv1.2 Null mouse A genomic phage clone containing the Kcna2 (Kv1.2 gene) was isolated from 129/Sv mouse liver library. A targetting construct with neomycin resistance was inserted between Kcna2 5'Xba I - Eag I and 3' Nco I - Xba I genomic fragment fig 1A. A thymidine kinase (TK) cassette was cloned into 3' portion of the targetting construct. Then the targeting construct was linearized and electroporated into AB-1 embryonic stem cells and were subjected to positive -negative selection in presence of 300 micromolar G418 and 200 nano Molar fialuridine. Then the doubly resistant clones were expanded. The Eco Rv resitriction endonuclease site in the Kcna2 open reading frame was removed following homologus recombination and positive clones were expanded and injected into C57BL/6J blastocysts.2 Breeding and Genotyping: The mutation was transfered into C3HeB/feJ mice by back cross to generate Kcna2 null mice (-/- mice). A chimeric founder was also crossed to C57BL/6J mice to establish a hybrid line and to provide mutant mice (-/- B6/129 mouse). DNA was isolated from tail veinclips and or ear punches and PCR amplified using primers to match open reading frame of Kcna2 and other to match neomycin resistance cassette indicating Kcna2 null genotype. futher confirmation was done via westernblots Fig 1c. Objectives of Generation Previous studies have shown that Kv1.1 (Kcna1 ) null mice exhibited seizure suceptibility and hyperexcitability in axons and medial nucleus of the trapezoid body (MNTB). Since both Kv1.2 and Kv1.1 are present as tetramers in juxtaparanodes in neuron the authors wanted to explore if kv1.2 null mice exhibit a similar phenotype and compare their seizure suseptability to the wild type mice. ObservationsLife Span: Kv1.2 null (-/-) mutations while being non embryonic lethal the mice had significantly reduced life span. (-/-) mice in average survived for 17 postnadal days while their litter mates (+/+) and (+/-) had normal life spans. Seizures: (-/-) mice were observed to undergo spontaneous seizures, while recovering from tonic extensions these mice exhibited myoclonic jerks and tremors followed by 5-20 min of relative immobility before recovery. the fatality rate during seizure was about 50% due to cessation of breathing. During Flurothyl induced seizures the (-/-) mice showed shortest mean latency for occurance of seizure followed by (+/-) and (+/+) mice. Action Potenials: (-/-) MNTB cells fired the smallest number of action poteintials and highest threshold current amplitude compared to the heterozygous or wildtype mouse.Fig 2.The (-/-) neurons also exhibited altered'' I - V ''relation and higher resting potentials and larger Ik currents. Conclusion In conclusion the authors suggest that the generation of genetically modified null mutant Kv1.2 mice enabled researchers to disect out the physiological role of Kv1.2 and differentiate it from Kv1.1 channels functionally since both the channels are very similar in sequence, expression and location. And also suggest that since Kv1 channels lacking Kv1.2 activated at abnormally negative potentials, it could explain why neurons with larger proportions of such channels had larger IKv1 and that if channel voltage dependence is determined by how many Kv1.2 subunits each contains,neurons might be able to fine-tune their excitability by adjusting the Kv1.1:Kv1.2 balance rather than altering Kv1 channel density 2. References 1 Chen, X., et al. (2010). "Structure of the full-length Shaker potassium channel Kv1.2 by normal-mode-based X-ray crystallographic refinement." Proceedings of the National Academy of Sciences 107(25): 11352-11357. 2 Brew, H. M., et al. (2007). "Seizures and reduced life span in mice lacking the potassium channel subunit Kv1.2, but hypoexcitability and enlarged Kv1 currents in auditory neurons." J Neurophysiol 98(3): 1501-1525 .